Field of the Invention
The present invention relates to a maintenance medium for primate pluripotent stem cells, comprising xylose.
Description of the Related Art
Pluripotent stem cells such as human ES cells (embryonic stem cells) and iPS cells (induced pluripotent stem cells) have been expected to be used and applied in various fields including regenerative medicine and cell therapy because they have the ability to be differentiated into various cells or tissues such as nerve cells and cardiac muscle cells, i.e., pluripotency. Pluripotent stem cells can be grown by culture after cell establishment. However, in pluripotent stem cells of primates including humans, it is not easy to grow the cells while maintaining the cell-specific properties, i.e., undifferentiation status and pluripotency (multipotency).
In order to grow human ES cells or human iPS cells while maintaining the undifferentiation status and the pluripotency, it is usually needed to culture these cells in coexistence with feeder cells, or to add substances for maintaining undifferentiation status, for example, ascorbic acid, basic fibroblast growth factor (bFGF), and transforming growth factor β-3 (TGF-β3), to a medium (refer to WO 2011/058558). However, the passage process in culture in coexistence with feeder cells is complicated, and many of substances for maintaining undifferentiation status are expensive. In addition, for human iPS cells, since it is known that the properties of the cells are changed when the passage number exceeds a certain number, it is preferable to make the passage number as small as possible.
Thus, cryopreservation is usually performed if the properties are maintained consistently, i.e., cells are preserved, for a medium and long term. However, primate, especially human, ES cells and iPS cells are known to be sensitive to freezing and thawing and to have a low viability after cryopreservation. Therefore, frequent cryopreservation cannot be performed for primate pluripotent stem cells.
Meanwhile, for human ES cells and iPS cells, it is difficult to prepare cells of different origin to a desired cell concentration during the same period because the growth rate is different by established cell line. For human ES cells and iPS cells, it is recommended to change a medium every day due to their fast metabolism, and the risk of contamination is high, placing a burden on researchers.
Therefore, in culture of primate pluripotent stem cells such as human ES cells and iPS cells, methods for simply maintaining and preserving pluripotent stem cells without cell passage while maintaining the properties of pluripotent stem cells other than cryopreservation are required. In other words, methods for temporarily controlling the proliferation of pluripotent stem cells without changing the properties of the cells are required.
Xylose is one of the constituent sugars of sugar chains, and plays an important role in vivo, such as in intercellular communication. Xylose is also known to be abundantly contained in woody biomass, and the expansion of its use as unused resources in various fields has been required. However, in the field of culture cells, xylose has not been used as a saccharide in a medium because cells are considered not to be able to use xylose as energy.
The present inventors previously confirmed the effects of various saccharides (e.g., glucose, xylose, galactose, and the like) on mouse ES cells, and reported the possibility that xylose maintains the undifferentiation status and its cell proliferation effects in mouse ES cells (Tadayuki Yokoyama et al., Effects of various sugars on cell proliferation and EB formation of mouse ES cells, The 7th Congress of the Japanese Society for Regenerative Medicine, Program and Abstract, Vol. 7, pp. 239, 2008; Tadayuki Yokoyama et al., Xylose maintains the undifferentiation status of mouse ES cells, The 8th Congress of the Japanese Society for Regenerative Medicine, Program and Abstract, Vol. 8, pp. 221, 2009; Sakiko Takizawa-Shirasawa et. al., The 6th International Niigata Symposium on Diet and Health, pp. 152, 2012.)
However, these literatures have no description of primate pluripotent stem cells, which is remarkably sensitive to changes in culture environment compared with mouse ES cells. To the inventors' knowledge, there are no reports on the use of xylose for cells other than mouse ES cells.
The present inventors found this time that in human iPS cells, when a medium in which glucose contained in a commercially available common medium for human iPS cells is replaced by xylose is used, the viability can be maintained and cell proliferation can be inhibited while the undifferentiation status and the pluripotency are maintained. In other words, human iPS cells were successfully maintained and preserved without cryopreservation and passage while the properties of human iPS cells are maintained. Despite the fact that saccharides other than glucose had usually been considered not to be able to be used as energy in cells, it was surprising that the viability could be maintained and the cells could be maintained at normal culture temperature for a long time over 1 week while the undifferentiation status and the pluripotency, which tend to be lost by culture environment degradation, are maintained. The present invention is based on these findings.